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Abstract:

The invention relates to isolated nucleic acids comprising mir-302 genes.
Also disclosed are expression vectors, host cells, and transgenic animals
containing the nucleic acids, and use of the nucleic acids to generate
ES-like cells.

Claims:

1. An isolated nucleic acid comprising one or more mir (microRNA)-302
genes operably linked to a regulatory sequence, wherein the regulatory
sequence controls the expression of the mir-302 genes.

2. The nucleic acid of claim 1, wherein the mir-302 genes are selected
from the group consisting of the mir-302a, mir-302b, mir-302c, and
mir-302d gene.

3. An isolated nucleic acid comprising a regulatory sequence operably
linked to a recombinant sequence encoding a contiguous transcript,
wherein the regulatory sequence controls the transcription of the
recombinant sequence, wherein the recombinant sequence comprises a first
gene encoding at least two exons flanking one intron, wherein the intron
comprises one or more mir-302s, and wherein the intron is spliced out of
the contiguous transcript of the recombinant sequence to allow the
mir-302s to interact with their targets in a cell.

4. The nucleic acid Of claim 3, wherein the mir-302s are selected from
the group consisting of mir-302a, mir-302b, mir-302c, and mir-302d.

5. The nucleic acid of claim 3, wherein the nucleic acid is transcribed
by a type II RNA polymerase.

6. The nucleic acid of claim 3, wherein the intron is spliced out of the
contiguous transcript of the recombinant sequence by a spliceosome.

7. The nucleic acid of claim 3, wherein the first gene is the RGFP (red
fluorescent HcRed1 chromoprotein) gene or a fragment thereof.

Description:

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application
Ser. Nos. 61/007,867, filed on Dec. 17, 2007, 61/060,416, filed on Jun
10, 2008, and 61/074,481, filed on Jun. 20, 2008, the contents of which
are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates in general to microRNAs. More
specifically, the invention provides isolated nucleic acids comprising
mir-302 genes, and expression vectors, host cells, and transgenic animals
containing such nucleic acids. The invention further provides methods of
generating ES-like cells using microRNAs.

BACKGROUND OF THE INVENTION

[0003] The concept of cancer stent cells indicates that transformed stem
cells within a tumor are able to self-renew and differentiate into a
heterogeneous tumor population (Reya et al., 2001). However, there is no
clear mechanism underlying such stem-cancer cell transformation or vice
versa. In the clinic, it is very frequent to observe that cancer
progression is generally associated with the poor differentiation (high
grade) of human tumor cells. Recent findings have also shown that poorly
differentiated tumors preferentially over-express genes normally enriched
in human embryonic stem (ES) cells, such as targets of Oct3/4, Sox2 and
Nanog transcription factors; nevertheless, the concurrent expression of
these transcription factors themselves is not often detected in the
poorly differentiated tumors (Ben-Porath et al., 2008). It is conceivable
that a different set of transcriptional regulators in place of the roles
of Oct3/4, Sox2 and Nanog may function in poorly differentiated tumor
cells to promote their "stemness" signatures. Therefore, finding this way
how stem cells hurdle these cancer-related transcriptional regulators may
shed light on breakthroughs in both cancer therapy and stem cell
generation.

[0004] The mir-302 family (mir302s) generally consists of four highly
homologous microRNA (miRNA) members, which are transcribed together as a
non-coding RNA cluster containing mir-302b, mir-302c, mir-302a, mir-302d,
and mir-367 from a 5' to 3' direction (Sub. et al., 2004). They are
expressed most abundantly in slow-growing human ES cells and the
expression quickly decreases alter cell differentiation and proliferation
(Suh et al., 2004). Given that miRNAs are characterized as small
inhibitory RNAs capable of suppressing the translation of target genes
with high complementarity (Bartel, D. P., 2004), mir-302s is a likely
candidate of zygotic inhibitors to prevent premature cell differentiation
during early embryonic development. As shown in the miRBase::Sequences
program at the website of microrna.sanger.ac.uk, mir-302s can target over
445 human genes and most of these targets are developmental signals
involving the initiation and/or facilitation of lineage-specific cell
differentiation during early human embryogenesis.

SUMMARY OF THE INVENTION

[0005] The present invention is based, at least in part, upon the
unexpected discovery that mir-302s reprogram human skin cancer cells into
a pluripotent ES-cell-like state.

[0006] Accordingly, in one aspect, the invention features an isolated
nucleic acid comprising one or more mir (microRNA)-302 genes operably
linked to a regulatory sequence. The regulatory sequence controls the
expression of the mir-302 genes.

[0007] In another aspect, the invention features an isolated nucleic acid
comprising a regulatory sequence operably linked to a recombinant
sequence encoding a contiguous transcript. The regulatory sequence
controls the transcription of the recombinant sequence. The recombinant
sequence comprises a first gene including at least two exons flanking one
intron. The intron comprises one or more mir-302s. The intron is spliced
out of the contiguous transcript of the recombinant sequence to allow the
mir-302s to interact with their targets in a cell.

[0008] A mir-302 gene may be the mir-302a, mir-302b, mir302c, or mir-302d
gene. Likewise, a mir-302 may be mir-302a, mir-302b, mir-302c, or
mir-302d. In some embodiments, a nucleic acid of the invention is
transcribed by a type II RNA polymerase. In some embodiments, an intron
of the invention is spliced out of the contiguous transcript of the
recombinant sequence by a spliceosome. Exemplary first genes include but
are not limited to the RGFP (red fluorescent HcRed1 chromoprotein) gene
or a fragment thereof.

[0009] The invention also provides an expression vector, a host cell, and
a transgenic animal comprising a nucleic acid of the invention.

[0010] In addition, the invention provides a method of generating ES
(embryonic stem)-like cells. The method comprises contacting non-ES-like
cells with a nucleic acid of the invention, thereby transforming the
non-ES-like cells into ES-like cells. In some embodiments, a method of
the invention further comprises inducing the ES-like cells to
differentiate into tissue cell types.

[0011] The non-ES-like cells may be cancer cells such as Colo or PC3
cells. Such cells, when transformed into ES-like cells, may form a
teratoma-like primordial tissue structure, fibroblasts, chondrocytes,
spermatogonia-like primordial cells, or neuronal cells.

[0012] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention pertains. In case of conflict,
the present document, including definitions, will control. The materials,
methods, and examples disclosed herein are illustrative only and not
intended to be limiting. Other features, objects, and advantages of the
invention will be apparent from the description and the accompanying
drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1. Strategy for generating transgenic mir-302s-expressing
mirPS cell lines, using retrovirus-based pLNCX2-rT-SpRNAi vector
transfection. A retroviral delivery approach was used to integrate a
cytomegalovirus (CMV) promoter-driven SpRNAi-RGFP transgene into the
tested cell genomes for steady expression of a manually redesigned
mir-302 pre-miRNA cluster (mir-302s). Mir-302s was placed in the intron
of the SpRNAi-RGFP transgene and generated as a part of the transgene
transcript RNA (pre-mRNA), containing RGFP protein-coding exons and
non-coding introns. The introns were spliced out of pre-mRNA and further
excised into small miRNA-like mir-302 molecules capable of triggering
targeted gene silencing, while the RGFP exons were ligated together to
form a mature mRNA for synthesis of a red fluorescent marker protein,
RGFP. The presence of RGFP served as an indicator for the expression and
processing of mir-302s.

[0014] FIG. 2. Reprogramming of human cancerous Colo and PC3 cells into
ES-like. mirPS cells with retrovirus-mediated transfection of mir-302s.
(A) Structure of a mir-302s-expressing SpRNAi-RGFP transgene located in
the XhoI/AflII cloning site of a cytomegalovirus (CMV) promoter-driven
pLNCX2 retroviral vector (Clontech), namely pLNCX2-rT-SpRNAi. (B)
Construct of the mir-302 pre-miRNA cluster (mir-302s), which was inserted
in the intron region of the SpRNAi-RGFP transgene. (C) Selection of mirPS
cells using FACS flow cytometry sorting with antibodies to RGFP and
Oct3/4. (D) Changes of morphologies and cell division rates in mirPS
cells. The first (left) and second (right) peaks of the DNA-density flow
cytometry charts represented the levels of resting G0/G1 and mitotic M
phase cell populations in the entire tested cell population,
respectively. The mir-gfp miRNA shared no homology to human genes. (E)
Loss of migration ability in mirPS-PC3 cells as compared to its original
PC3 cells. (F) Formation of embryoid bodies derived from mirPS cells and
their differentiation into neuron-like primordial cells with Tuj1 and
ABCA2 marker expression.

[0015] FIG. 3. Teratoma-like primordial tissues derived from the mirPS EB
implants in the uterus or peritoneal cavity of female pseudopregnant
immunocompromised SCID-beige mice. These differentiated tissues included
all three embryonic germ layers--ectoderm, mesoderm and endoderm, as
determined by their distinct cell morphologies after hematoxylin and
eosin (H&E) staining. Photographs were taken with the Nikon TE2000
microscopic system at 200× magnification.

[0017] FIG. 5. Genome-wide gene expression analyses among Colo, mirPS-Colo
and human ES WA01-H1 (H1) and WA09-H9 (H9) cells. (A) Comparison of
altered gene expression patterns using Human genome GeneChip U133A&B and
plus 2.0 arrays (Affymetrix), showing high similarity between mirPS-Colo
and H1 (89%) as well as H9 (86%), but not original Colo (53%) cells.
(B)-(E) Functional clustering of microarray-identified differentially
expressed genes, demonstrating that a significant increase of ES cell
markers (B) and a marked decrease of melanoma oncogenes (C),
developmental signals (D), and mir-302s-targeted cell proliferation and
DNA methylation genes (E) were detected in mirPS cells, which highly
resembled those in H1 and H9 cells (n=4, p<0.01). Any signal showing
above the level 23,000 of total 36,535 (in red) was considered to be a
positive call in the gene expression list.

[0018] FIG. 6. Pluripotency of mirPS cells. Treatments of DHT, TGF-β1
and BMP4, respectively from top to bottom, induced the mirPS-Colo cell
differentiation into spermatogonia-like (A-E), fibroblast-like (F-J) and
chondrocyte-like (K-O) primordial cells, in immunocompromised mice ex
vivo. The use of immunocompromised nude mice was to provide an in vivo
environment mimicking transplantation therapy. Microscopic photographs
shown from left to right indicated hematoxylin staining with differential
interference contrast (A, F, K), bright field labeled with transgenic
mir-302 marker RGFP (red) (B, G, L), immunostaining of the first tissue
marker labeled with 4,6-diamidino-2-phenylindole (blue: DAPI) (C, H, M),
immunostaining of the second tissue marker labeled with fluorescein
(green EGFP) (D, I, N), and merge of all three fluorescent markers (E, J,
O). Small windows in the RGFP-bright fields showed the morphologies of
differentiated mirPS cells at high magnification (600 ×).

[0019]FIG. 7. List of differentially enriched miRNAs in mirPS-Colo cells.
The expression of mir-302 familial members, including mir-302a, mir-302b,
mir-302 c and mir-302d, were all highly elevated in mirPS-Colo (sample B)
as compared to the original Colo (sampler A) cells. Concurrently, one of
the reverse mir-302, mir-302a*, was also markedly expressed.

[0020] FIG. 8. Transgenic integration of the mir-302s-expressing
SpRNAi-RGFP transgene in mirPS cells. (A) Quantitative PCR analyses of
the genomic DNAs isolated from mirPS cells, showing that all tested mirPS
cells carried only one or two copies of the transgene, whereas no
transgene was detected in the original Colo and PC3 cells. (B)
Fluorescent in situ hybridization (FISH) detection of genomic transgene
insertion. Approximately 75% of mirPSPC3 and 17% of mirPS-Colo cells
contained one transgene insert in their genomes, while the others
contained two inserts, but no more than three. Many of these two inserts
were concomitantly placed to each other, an event frequently observed in
high-titer retroviral infection. Such restricted transgene insertion
indicates that the mir-302 expression level may affect the survival of
the mirPS cells.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates to the utilities of microRNAs in
generating ES-like cells. As described in detail below, to test the
function of mir-302s, a retroviral Pol-II-based intronic miRNA expression
system was developed, namely pLNCX2-rT-SpRNAi (FIG. 1), and successfully
used to generate several transgenic miRNA-expressing cell lines and
animals (Lin and Ying, 2006; Lin et al., 2006). The same transgenic
approach has also been used to generate gene-knockout mice for human
disease research (Xia et al., 2006). Intronic miRNA expression is a
prevalent event in mammals because approximately 50% of mammalian miRNAs
are encoded within the introns of protein-coding genes (Rodriguez et al.,
2004). These miRNAs are transcribed by type II RNA polymerases. (Pol II)
and excised by spliceosomes and other RNaseIII endonucleases to form
mature miRNAs (Lin et al., 2003; Danin-Kreiselman et al., 2008). However,
Drosha may not be required for this process (Ruby et al., 2007). The
composition of this mir-302s-expressing pLNCX2-rT-SpRNAi vector is shown
in FIG. 2A. Using this vector-based transaction strategy, two
mir-302s-expressing mirPS cell lines were generated, namely mirPS-Colo
and mirPSPC3, derived from human melanoma Colo and prostate cancer PC3
cellar, respectively, and their ES-like cell renewal and pluripotent
properties confirmed.

[0022] Accordingly, the invention provides an isolated nucleic acid
comprising one or more mir-302 genes operably linked to a regulatory
sequence, wherein the regulatory sequence controls the expression of the
mir-302 genes.

[0023] The term "isolated nucleic acid" includes nucleic acid molecules
that are separated from other nucleic acid molecules that are present in
the natural source of the nucleic acid. For example, with respect to
genomic DNA, the term "isolated" includes nucleic acid molecules that are
separated from the chromosome with which the genomic DNA is naturally
associated. Preferably, an "isolated" nucleic acid is free of sequences
that naturally flank the nucleic acid (i.e., sequences located at the 5'
and/or 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. Moreover, an "isolated" nucleic
acid molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized.

[0025] Genes and RNAs described herein can be replaced by their
functionally equivalent fragments or homologs (e.g., with at least 50%,
60%, 70%, 80%, or 90% sequence homology). In particular, mir-302a,
mir-302b, mir-302c, and mir-302d genes and RNAs described herein may be
replaced with other genes and RNAs with similar functions such as
mir-302a*, mir-302b*, mir-302c*, mir-367, mir-93, mir-371, mir-372,
mir-373, mir-520, and the like.

[0026] The term "regulatory sequence" includes promoters, enhancers and
other expression control elements (e.g., polyadenylation signals).
Regulatory sequences include those that direct constitutive expression of
a nucleotide sequence, as well as tissue-specific regulatory and/or
inducible sequences.

[0027] Another isolated nucleic acid of the invention comprises a
regulatory sequence operably linked to a recombinant sequence encoding a
contiguous transcript. The regulatory sequence controls the transcription
of the recombinant sequence. The recombinant sequence comprises a first
gene encoding at least two exons flanking one intron. The intron
comprises one or more mir-302s. The intron is spliced out of the
contiguous transcript of the recombinant sequence to allow the mir-302s
to interact with their targets in a cell.

[0028] As used herein, a "recombinant" sequence refers to a sequence that
does not occur in nature.

[0029] Preferably, the nucleic acid is transcribed by a type II RNA
polymerase, and the intron is spliced out of the contiguous transcript of
the recombinant sequence by a spliceosome. Regulatory sequences such as
promoters for type II RNA polymerases are generally known in the art.
See, for example, Smale and Kadonaga (2008) Annu Rev Biochem 72:449-479.
The cis and trans elements-required for spliceosomal splicing are also
known to a skilled artisan. See, for example, Lewin B., Genes, Seventh
Edition, Oxford University press, page 689, 2000. Therefore, one skilled
in the art may construct a nucleic acid of the invention using
recombinant DNA techniques or chemical synthesis.

[0030] To monitor the transcription of the recombinant sequence and
subsequent processing of the transcript, a detectable marker gene may be
used as the first gene. For example, as described in detail below, when
the RGFP gene is employed as the first gene, the presence of the RGFP
protein serves as an indicator for the transcription of the recombinant
sequence and the processing of the transcript.

[0031] A nucleic acid of the invention can be included in a vector,
preferably an expression vector. As used herein, the term "vector" refers
to a nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked and can include a plasmid, cosmid or viral
vector. The vector can be capable of autonomous replication or it can
integrate into a host DNA. Viral vectors include, e.g., replication
defective retroviruses, adenoviruses and adeno-associated viruses.

[0032] A vector can include a nucleic acid of the invention in a form
suitable for the expression of the nucleic acid in a host cell. The
design of the expression vector can depend on such factors as the choice
of the host cell to be transformed, the level of mir-302 gene expression
desired, and the like. The expression vectors of the invention can be
introduced into host cells to thereby produce mir-302s.

[0033] The expression vectors of the invention can be designed for the
expression of the mir-302 genes in a variety of cells such as insect
cells (e.g., using baculovirus expression vectors), yeast cells, or
mammalian cells. Suitable host cells are discussed further in Goeddel,
Gene Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the expression vector can be
transcribed in vitro, for example using T7 promoter regulatory sequences
and T7 polymerase.

[0034] When used in mammalian cells, the expression vector's control
functions are often provided by viral regulatory elements. For example,
commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40.

[0036] The invention further provides a host cell that includes a nucleic
acid of the invention. The nucleic acid may be within an expression
vector or homologously recombine into a specific site of the host cell's
genome. The terms "host cell" refers not only to the particular subject
cell but to the progeny or potential progeny of such a cell.

[0037] A nucleic acid or vector of the invention can be introduced into
host cells via conventional transformation or transfection techniques. As
used herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing a
foreign nucleic acid (e.g., DNA) into a host cell, including calcium
phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated
transfection, lipofection, or electroporation.

[0038] A host cell of the invention can be used to produce (i.e., express)
mir-302s. Accordingly, the invention further provides methods for
producing mir-302s using the host cells of the invention. In one
embodiment, the method includes culturing the host cells of the invention
in a suitable medium such that mir-302s are produced.

[0039] The invention additionally features non-human transgenic animals
containing a nucleic acid of the invention. Such animals are useful for
studying the function and/or activity of mir-302s. As used herein, a
"transgenic animal" is a non-human animal, preferably a mammal, more
preferably a rodent such as a rat or mouse, in which one or more of the
cells of the animal include a transgene containing a nucleic acid of the
invention. Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A
transgene is an exogenous DNA, which preferably is integrated into the
genome of the cells of a transgenic animal. A transgene can direct the
expression of an encoded gene product in one or more cell types or
tissues of the transgenic animal.

[0040] Intronic sequences and polyadenylation signals can also be included
in the transgene to increase the efficiency of the expression of the
transgene. A tissue-specific regulatory sequence can be operably linked
to a transgene of the invention to direct the expression of mir-302s to
particular cells. A transgenic founder animal can be identified based
upon the presence of a transgene in its genome and/or expression of
mir-302s in tissues or cells of the animal. A transgenic founder animal
can then be used to breed additional animals carrying the transgene.
Moreover, transgenic animals carrying a transgene can further be bred to
other transgenic animals carrying other transgenes.

[0041] The invention also includes a population of cells from a transgenic
animal, as discussed herein.

[0042] A method of generating ES-like cells is within the invention. The
method involves contacting non-ES-like cells with a nucleic acid of the
invention, thereby transforming the non-ES-like cells into ES-like cells.
This method may be practiced in vivo, in vitro, or ex vivo.

[0043] The term "ES-like cells" refers to cells derived from adult or
mature, non-pluripotent cells but have many or all of the characteristics
of embryonic stem cells. ES-like cells may be identified using protocols
well known in the art. For example, as described in detail below, ES-like
cells may be identified using antibodies to ES markers such as Oct3/4,
SSEA-3, SSEA-4, Sox2 and Nanog, by detecting a slow rate of cell cycle,
changes in cell morphology, loss of ability to migrate, genomic
demethylation, or inhibition of cell cycle checkpoint genes (e.g., CDK2
and cyclin D1 and D2) and DNA methylation facilitator genes (e.g., MECP2
and MECP1 component p66), or by detecting the pluripotency of the cells.

[0044] When a nucleic acid of the invention is introduced into non-ES-like
cells, mir-302s are produced from the nucleic acid and interact with
their targets in the cells. Most of these target genes are developmental
signals involving the initiation and/or facilitation of lineage-specific
cell differentiation during early embryogenesis. Thus, mir-302s are key
factors essential for ES cell maintenance.

[0045] As described in detail below, cancer cells can be reprogrammed by
mir-302s into a more ES-like state. Accordingly, a nucleic acid of the
invention may be used in a cancer therapy. A treatment method of the
invention involves administering an effective amount of a nucleic acid of
the invention to a subject suffering from cancer.

[0046] As used herein, a "subject" refers to a human or animal, including
all mammals such as primates (particularly higher primates), sheep, dog,
rodents (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, and
cow. In a preferred embodiment, the subject is a human. In another
embodiment, the subject is an experimental animal or animal suitable as a
disease model.

[0047] A subject to be treated may be identified in the judgment of the
subject or a health pare professional, which can be subjective (e.g.,
opinion) or objective (e.g., reached by detecting a cancer marker in the
subject).

[0048] A "treatment" is defined as administration of a substance to a
subject with the purpose to cure, alleviate, relieve, remedy, prevent, or
ameliorate a disorder, symptoms of the disorder, a disease state
secondary to the disorder, or predisposition toward the disorder.

[0049] An "effective amount" is an amount of a compound that is capable of
producing a medically desirable result in a treated subject. The
medically desirable result may be objective (i.e., measurable, by some
test or marker) or subjective (i.e., subject gives an indication of or
feels an effect).

[0050] For treatment of cancer, a compound is preferably delivered
directly to tumor cells, e.g., to a tumor or a tumor bed following
surgical excision of the tumor, in order to treat any remaining tumor
cells.

[0051] Nucleic acids can be delivered to target cells by, for example, the
use of polymeric, biodegradable microparticle or microcapsule devices
known in the art. Another way to achieve uptake of nucleic acids is to
use liposomes, prepared by standard methods. The nucleic acids can be
incorporated alone into these delivery vehicles or co-incorporated with
tissue-specific or tumor-specific antibodies. Alternatively, one can
prepare a molecular conjugate composed of a nucleic acid attached to
poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to
a ligand that can bind to a receptor on target cells. "Naked DNA" (i.e.,
without a delivery vehicle) can also be delivered to an intramuscular,
intradermal, or subcutaneous site. Generally, preferred dosage for
administration of nucleic acids is from approximately 106 to
1012 copies of the nucleic acid molecule.

[0052] A nucleic acid of the invention can be incorporated into
pharmaceutical compositions. Such compositions typically include the
therapeutic compounds and pharmaceutically acceptable carriers.
"Pharmaceutically acceptable carriers" include solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with pharmaceutical
administration.

[0054] It is advantageous to formulate oral or parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage.
"Dosage unit form," as used herein, refers to physically discrete units
suited as unitary dosages for the subject to be treated, each unit
containing a predetermined quantity of an active compound calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical carrier.

[0055] The dosage required for treating a subject depends on the choice of
the route of administration, the nature of the formulation, the nature of
the subject's illness, the subject's size, weight, surface area, age, and
sex, other drugs being administered, and the judgment of the attending
physician. Suitable dosages are in the range of 0.01-100.0 mg/kg. Wide
variations in the needed dosage are to be expected in view of the variety
of compounds available and the different efficiencies of various routes
of administration. For example, oral administration would, be expected to
require higher dosages than administration by intravenous injection.
Variations in these dosage levels can be adjusted using standard
empirical routines for optimization as is well understood in the art.
Encapsulation of the compound in a suitable delivery vehicle (e.g.,
polymeric microparticles or implantable devices) may increase the
efficiency of delivery, particularly for oral delivery.

[0056] Moreover, a method of the invention may further comprise inducing
the ES-like cells to differentiate into tissue cell types. Through in
vitro manipulations with different factors and/or hormones, the ES-like
cells can differentiate into the three embryonic germ layers (ectoderm,
mesoderm and definitive endoderm)--the founders of all adult tissues.
Absent any treatment, xenograft implantation of embryoid bodies derived
from the ES-like cells into an animal or human can form various tissue
structures. For example, as described in detail below, after in vitro
treatments of various growth factors and/or hormones, the mirPS-Colo
cells differentiate into several tissue cell types ex vivo, including
fibroblasts, chondrocytes and spermatogonia-like primordial cells.
Xenograft implantation of the mirPS-Colo-derived embryoid bodies into the
uterus or peritoneal cavity of female pseudopregnant immunocompromised
SCID-beige mice forms teratoma-like primordial tissue structures.

[0057] The following examples are intended to illustrate, but not to
limit, the scope of the invention. While such examples are typical of
those that might be used, other procedures known to those skilled in the
art may alternatively be utilized. Indeed, those of ordinary skill in the
art can readily envision and produce further embodiments, based on the
teachings herein, without undue experimentation.

[0059] Stem cell renewal differs from cancer cell growth in its highly
self-regulated cell division pattern. The mir-302 microRNA family
(mir-302s) is expressed most abundantly in slow-growing human embryonic
stem (ES) cells, and the expression quickly decreases after cell
differentiation and proliferation. Therefore, mir-302s were investigated
as the key factors essential for maintenance of ES cell renewal and
pluripotency in this study. The Pol II-based intronic microRNA (miRNA)
expression system was used to transgenically transfect the mir-302s into
several human cancer cell lines. The mir-302-transfected cells, namely
miRNA-induced pluripotent stem (mirPS) cells, not only expressed many key
ES cell markers, such as Oct3/4, SSEA-3, SSEA-4, Sox2 and Nanog, but also
had a highly demethylated genome similar to a reprogrammed zygotic
genome. Microarray analyses further revealed that genome-wide gene
expression patterns between the mirPS and human ES H1 and H9 cells shared
over 86% similarity. Using molecular guidance in vitro, these mirPS cells
could differentiate into distinct tissue cell types, such as neuron-,
chondrocyte-, fibroblast- and spermatogonia-like primordial cells. Based
on these findings, we conclude, that mir-302s not only function to
reprogram cancer cells into an ES-like pluripotent state, but also to
maintain this state under a feeder-free cultural condition, which offers
a great opportunity for therapeutic intervention.

[0060] After the pLNCX2-rT-SpRNAi retroviral transfection with a
pre-designed mir-302 pre-miRNA cluster transgene (FIG. 2B), approximately
95%-98% of the transfected cells underwent apoptosis with the remaining
2%-5% of the cells transformed into ES-like mirPS cells. The transfection
rates of mir-302s info Colo and PC3 cells were 99.8% and 99.4%,
respectively, as determined by FACS flow cytometry sorting with mir-302
maker RGFP and ES marker Oct3/4 antibodies (FIG. 2C). These mirPS cells
could grow in either DMEM/F12 of RPMI 1640/B27 medium supplemented with
10% charcoal-stripped FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, 5
ng/ml activin, 5 ng/ml noggin, 3 ng/ml bFGF and an equal mixture of 0.5
μM Y27632 and 0.5 μM GSK-3 inhibitor XV, at 37° C. under 5%
CO2. Under this feeder-free cultural condition, the average cell
cycle of the mirPS cells was about 20-24 hours, indicating a very slow
cell renewal rate as compared to their cancerous counterparts. The flow
cytometry analysis comparing DNA content to cell cycle stages showed a
greater than 67% reduction in the mirPS mitotic cell population (FIG.
2D). The mitotic cell population (M phase) was decreased from 36.5% to
11.5% in mirPS-Colo and from 38.4% to 12.6% in mirPS-PC3 cells, whereas
no change was found in the control cells transfected with either an empty
pLNCX2-rT-SpRNAi vector (cell+vector) or a vector encoding an off-target
mir-gfp pre-miRNA construct (cell+mirgfp). Accordingly, the mirPS cell
morphology (lower panels) was changed from a spindle- or asterisk-like
form to a more rounded shape, indicating that the mirPS cells may have
lost their ability to migrate. As shown in FIG. 2E, metastatic PC3 cells
quickly migrated over time, whereas mirPS-PC3 cells remained stationary.
Thus, such transgenic mir-302s expression is sufficient to transform
human cancer cells into a more ES-like cell morphology and rate of cell
division, indicating a very beneficial use in cancer therapy.

[0061] MirPS cells were able to form compact colonies reminiscent of
embryoid bodies (EB) derived from human ES cells (FIG. 2F). When
dissociated with collagenase IV and then cultivated in RPMI 1640 medium
supplemented with 10% FBS, many of these EB-like cells differentiated
into neuronal cells based on the presence of positive neuronal markers
Tuj1 and ABCA2. We also noted that mirPS-PC3 EB cells could only
differentiate into neuronal cell types, while mirPS-Colo EB cells formed
teratoma-like primordial tissue structures in immunocompromised
SCID-beige mice (FIG. 3), suggesting that different cancerous stem cells
may have different pluripotent potentials. In view of the broad
pluripotency in mirPS-Colo cells, we therefore evaluated the correlation
between mir-302 and ES marker expression within the mirPS-Colo cells. As
shown in FIG. 4A and FIG. 7, miRNA microarray analyses demonstrated that
the expression rates of all mir-302s were significantly increased over
eight folds in the mirPS-Colo cells. Since the four mir-302 members share
very high homology and target almost the same cellular genes, this result
indicates that the overall gene silencing effects of mir-302s may
increase over thirty folds in the mirPS cells. Genomic PCR and
fluorescent in situ hybridization assays further revealed that all mirPS
cells carried either one or two copies of the mir-302s transgene (FIG.
8). Thus, the concentration of mir-302s may affect both pluripotency and
survival of the mirPS cells.

Identification of Human ES Cell Markers

[0062] Consistent with the elevation of mir-302s expression, many human ES
cell markers were strongly detected in the mirPS cell, as determined by
Western blot analyses (FIG. 4B). These ES markets, including Oct3/4,
SSEA-3, SSEA-4, Sox2 and Nanog, were barely detected in both the original
Colo cancer cells and the cells transfected with an empty
pLNCX2-rT-SpRNAi vector (Colo+vector), a vector expressing mir-gfp miRNA
(Colo+mir-gfp), or a vector expressing nonhomologous mir-434-5p pre-miRNA
(Colo+mir-434-5p). Ben-Porath et al. (2008) have recently reported that
many central regulators of ES identity, such as Oct3/4, Sox2 and Nanog,
were not broadly expressed in high-grade cancers. Given that the
concurrent expression of Oct3/4, SSEA-3 and SSEA-4 is a key determinant
of human ES cell identity (Thomson et al., 1998), activation of these ES
marker genes in mirPS-Colo cells indicates that mir-302s must provide
certain "stemness" in order to reprogram the cancerous Colo cells into a
human ES-cell-like state. As the miRBase::Sequences program at the
website of microrna.sanger.ac.uk has predicted that methyl-CpG binding
proteins, MECP2 and MECP1 component p66, are both strong targets of
mir-302s, suppression of nuclear DNA methylation may be a mechanism
underlying this reprogramming process.

Assessment of Reprogramming-Related Genomic Demethylation

[0063] Change of epigenetic modification underlines another unique feature
of ES cells, particularly genomic demethylation (Hochedlinger and
Jaenisch, 2006). In order to reprogram a cell into its ES state, many
embryonic genes need to be re-activated by DNA demethylation, such as
Oct3/4. To assess this effect in the mirPS cells, we first performed a
whole genome digestion with HpaII, a restriction enzyme that is sensitive
to CpG methylation and cleaves only an unmethylated CCGG rather than
methylated CCGG site. FIG. 4c shows that the digested DNA fragments from
control Colo cells are over twice as large as those from the mirPS-Colo
cells, indicating that the mirPS genome is highly demethylated. Further
assessment in the Oct3/4 gene promoter region was performed using
bisulfite PCR and genomic DNA sequencing (Takahashi and Yamanaka, 2006),
which converted all unmethylated cytosines to uracils. Because
unmethylated ACGT sites were also changed into AUGT sites by bisulfite,
the digestion of mixed ACGT-cutting restriction enzymes failed to cleave
this isolated region from the mirPS cells (FIG. 4D). The detailed
demethylation maps shown by the bisulfite sequencing further demonstrated
that over 90% methylation sties of the Oct3/4 gene promoter region were
lost in the mirPS cells (FIG. 4E), suggesting that an epigenetic
reprogramming process did occur to re-activate the Oct3/4 expression.
Such epigenetic reprogramming is correlated to mir-302s expression
because no DNA demethylation was found in cells transfected with an empty
mir-302-free vector as compared to control cancer cells.

Microarray Analysis of Genome-Wide Gene Expression Profiles

[0064] Genome-wide gene profiling was required to determine the genetic
alterations associated with this mir-302-mediated reprogramming event.
Microarray analysis was used to screen changes in genome-wide gene
expression patterns in cells before and after the mir-302s transfection,
as well as between the mirPS cells and human ES H1 and H9 cells. The
changes in over 47,000 human gene expression patterns were assessed using
Affymetrix gene microarrays (GeneChip U133A&B and U133 plus 2.0 arrays).
We first duplicated the microarray tests using the same mirPS sample and
selected two hundred most variable genes (white dots) from one of the
tests for further comparison. As shown in FIG. 5A, the changes of these
selected gene expressions were all less than one fold in the duplicated
tests, indicating that the background variation was limited. Based on the
scattering patterns of all microarray-identified genes, we then
calculated the correlation coefficiency (CC) between the results of two
compared transcriptome libraries. A CC rate was given to show the
percentage of similarity in the genome-wide gene expression patterns with
a threshold of only one-fold change. Under such stringent CC rate
definition, we found that the gene expression patterns of mirPS-Colo
cells were very similar to those of ES H1 (89%) and H9 (86%) cells,
whereas only a low 53% CC rate was shown between Colo and mirPS-Colo
cells. This strong genetic correlation between human ES and mirPS cells
suggests that mir-302s may alter thousands of cellular gene expressions,
which are involved in the reprogramming process of a cancer cell into an
ES-like mirPS cell. For example, the elevation of many ES gene
expressions and shutdown of numerous oncogenic, developmental and
mir-302-targeted cell, cycle-related genes were consistently and
concurrently observed in the mirPS and human ES results, as shown in
FIGS. 5B-E.

[0065] In FIG. 5E, we noted that cell-cycle checkpoint genes, i.e., CDK2,
cyclin D1 and D2, and DNA methylation facilitator, i.e., MECP2 and MECP1
component p66, were all confirmed to be strong targets for mir-302s. It
is known that cyclin E-dependent CDK2 is required for the entry of
S-phase cell cycle and inhibition of CDK2 results in G1-phase checkpoint
arrest, whereas cyclin D1 can override G1-phase arrest in response to DNA
damage (Sherr and Roberts, 2008). Based on this principle, the
suppression of both CDK2 and cyclin D1 in mirPS cells revealed a fact
that the cell cycle of mir-302s-transfected cancer cells could reach a
very slow cell division rate as shown in FIG. 2D. The result of such
cancer-stem cell cycle transition provides a significant benefit in
cancer therapy. In addition, the suppression of MECP2 and MECP1/p66
activities was consistent with the results of FIGS. 4C-E, which indicated
the epigenetic reprogramming of malignant cancer cells into benign mirPS
cells. It is conceivable that the mirPS cells so obtained from patients
may be further used to repair the cancer damages.

In Vitro Molecular Guidance of mirPS Cell Differentiation

[0066] Pluripotency defines the most important characteristic of an ES
cell. Through in vitro manipulations with different factors and/or
hormones, human ES cells can differentiate into the three embryonic germ
layers (ectoderm, mesoderm and definitive endoderm)--the founders of all
adult tissues. In the absence of any treatment, xenograft implantation of
the mirPS-Colo-derived embryoid bodies into the uterus or peritoneal
cavity of female pseudopregnant immunocompromised SCID-beige mice formed
teratoma-like primordial tissue structures (FIG. 8). The growth of these
teratoma-like structures was terminated approximately 2.5-week
post-implantation. It seems that there is a self-regulation mechanism
limiting the random growth of these mirPS/EB cells in vivo. However,
using in vitro treatments of various growth factors and/or hormones, we
could direct the mirPS-Colo cells to differentiate into several tissue
cell types ex vivo, including fibroblasts (FIGS. 6A-E), chondrocytes
(FIGS. 6F-J) and spermatogonia-like (FIGS. 6K-O) primordial cells. The
protocols for these in vitro conditions and xenotransplantation methods
are provided in the Materials and Methods. Markers for the special tissue
lineages were also identified with immunohistochemical detection, showing
germ line-specific Dazla and EE2, fibroblast-specific atlastin1 and type
I pro-collagen (COL1A1), and chondrocyte-specific tropoelastin and type
II pro-collagen (COL2A1), respectively. These findings confirmed the
pluripotency of the mirPS cells. It is conceivable that many more tissue
cell types may be induced from these mirPS cells, using different
molecular interventions.

DISCUSSION

[0067] Ever since the first isolation of human ES cell line from human
blastocysts (Thomson et al., 1998), there were concerns about destruction
of human embryos, contamination of feeder cell antigens, and formation of
teratomas. Recent reports on induced pluripotent stem (iPS) cells have
opened up a new avenue for generating ES-like pluripotent cells directly
from adult body cells, bypassing the use of human embryos as starting
materials (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). Using
retroviral delivery of four transcription factor genes (i.e., either
Oct4-Sox2-c-Myc-Klf4 or Oct4-Sox2-Nanog-Lin28) into mouse embryonic
fibroblasts, the iPS cells so obtained were similar in many genetic and
behavioral properties to mouse embryonic stem cells (Okita et al., 2007;
Wernig et al., 2007). Additional iPS cell lines have continued to be
developed from human embryonic fibroblasts and primary dermal fibroblast
cultures using a similar approach (Yu et al., 2007; Park et al., 2008).
Yet, there are two problems emerging from the process of iPS cell
generation; one is the use of retroviral transgenes and the other the use
of oncogenes (e.g., c-Myc and Klf4). Retroviral transfection is the only
effective means to simultaneously and transgenically deliver the four
full-length genes into a targeted somatic cell, whereas the random
insertion of retroviral vectors into the transfected cell genome may also
affect other non-targeted genes and produce unexpected results. This is
problematic because simultaneous delivery of four large transgenes into
one single cell is difficult to control, particularly when one or more of
the genes are oncogenes.

[0068] Unlike the previous iPS cell technology, each member of the mir-302
family is able to simultaneously regulate over 445 cellular genes and
they all share almost the same target genes based on the databases from
the miRBase::Sequences program at the website of microrna.sanger.ac.uk.
Many of the mir-302 targeted genes are active developmental signals
involved in initiation or facilitation of lineage-specific, cell
differentiation during early embryonic development. Thus, the function of
mir-302s is more likely to attenuate the global production of
developmental signals rather than to create transcriptional stimulation
on certain embryonic signaling pathways. By inhibiting the cellular genes
essential for embryonic development and cell differentiation, mir-302s is
not only able to reprogram differentiated cancer cells into ES-like
pluripotent stem cells but also to maintain their pluripotency and
renewal under a feeder-free culture condition. Nevertheless, mir-302s may
not be the only miRNA family involved in this mechanism because their
target genes are also redundantly silenced by the group of mir-93,
mir-367, mir-371, mir-372, mir-373 and mir-520 in human ES cells.
Learning why these target genes must be simultaneously silenced during
the reprogramming process of cancer-ES cell transformation may shed light
on the mechanism underlying this miRNA-mediated gene silencing effect on
ES cell maintenance and renewal.

[0069] Utilization of intronic mir-302 transfection provides a safe and
powerful new tool for human ES-like pluripotent cell generation,
particularly derived from cancerous and primarily cultured somatic cells.
Because the intronic miRNA pathway is tightly regulated by multiple
intracellular surveillance systems, such as mRNA transcription, RNA
splicing, exosomal digestion and nonsense-mediated decay (NMD)
mechanisms, it is considered to be much more effective, specific and safe
than the siRNA/shRNA pathway (Lin et al., 2008). Advantageously, there
are three breakthroughs in this mir-302-induced mirPS cell generation
method. First, the transfection of a single mir-302s-expressing transgene
offers a very simple, efficient and safe method for generating ES-like
pluripotent stem cells, preventing the tedious retroviral insertion of
all four large transcription factor genes into one single cell as
demonstrated in the previous iPS methods. Second, because the size of the
mir-302s-expressing transgene is just about 1 kilo-bases, the
transfection efficiency is extremely high (almost 100%) and the selection
of positive mirPS cells can be easily carried out by passing once through
FACS flow cytometry, which is a very time-saving process. Third, the
transfection process can be completed under a feeder-free condition
without the risk of feeder antigen contamination and the mirPS cells so
obtained can continue to grow in a feeder-free cultural condition.
Fourth, no oncogene is used in this mirPS cell generation process.
Lastly, we may use homologous DNA insertion in place of retroviral
transfection to deliver the mir-302s-expressing transgene into a
specific, desired region of the cell genome, preventing the risk of
random insertion. Given that these advantages have solved most of the
problems found in current stem cell research, these mirPS cells are
useful for transplantation and cell therapies.

Incorporation of the SpRNAi-RGFP Transgene into a pLNCX2-rT-SpRNAi
Retroviral Vector

[0072] We modified a VSV-G-positive pantropic retroviral vector, namely
pLNCX2-rT, to transgenically deliver the mir-302-encoded SpRNAi-RGFP
transgene (Lin et al., 2006), The pLNCX2-rT vector was derived from a
modified pseudotype Moloney Murine Leukemia virus, pLNCX2 (Clontech, Palo
Alto, Calif.). As shown in FIG. 1, we first incorporated the SpRNAi-RGFP
transgene into the XhoI/AflII restriction site of the pLNCX2-rT vector
and then inserted the mir-302 pre-miRNA cluster construct into the
intronic insertion site. (PvuI/MluI restriction site) of the SpRNAi-RGFP
transgene, so as to form a retroviral pLNCX2-rT-SpRNAi transgene vector
capable of transgenically expressing mir-302s. In the experiments, we
mixed an equal amount (1:1) of the SpRNAi-RGFP transgene and the
pLNCX2-rT retroviral vector, and then digested the mixture with XhoI and
AflII restriction enzymes at 37° C. for 4 hours. The digested
mixture was collected with a microcon-30 filter and ligated together with
T4 DNA ligase (20 U, Roche Biochemicals, Indianapolis, Ind.) at
12° C. for 12 hours. Following the same protocol except using
PvuI/MluI digestion, we incorporated the mir-302 pre-miRNA cluster into
the PvuI/MluI restriction site of the pLNCX2-rT-SpRNAi vector. The
pre-miRNA-inserted pLNCX2-rT-SpRNAi vector was propagated E. coli
DH5α LB cultures containing 100 μg/ml ampicillin (Sigma) and
purified with a QIAprep spin miniprep kit (Qiagen), following the
manufacturer's suggestion. For viral production, the pLNCX2-rT-SpRNAi
vector was co-transfected with an equal amount of pVSV-G vector into
GP2-293 packaging cells (Clontech) to produce infectious, but not
replicable, pantropic retroviruses. GP2-293 cells were grown in phenol
red-free DMEM medium supplemented with 10% FBS, 4 mM L-glutamine and 1 mM
sodium pyruvate. The cotransfection was carried out with a FuGene 6
reagent (Roche), following the manufacturer's suggestion.

MirPS Cell Generation Using the pLNCX2-rT-SpRNAi Transgene Vector

[0073] High titer viruses were released in the DMEM medium of the GP2-293
cell cultures approximately 36-48 hours after the co-transfection of
pVSV-G and pLNCX2-rT-SpRNAi vectors. The viral titer was measured to be
over multiplicity of infection (MOI) 30 before call transfection,
following the protocol of a retro-X qRT-PCR titration kit (Clontech).
Then, we transferred the high titer virus medium to the Colo or PC3 cell
cultures and incubated the cells for 12 hours at 37° C. under 5%
CO2. After that, fresh mirPS cell culture medium was added in place
of the virus medium and replaced every three days. Positively transfected
cells were isolated and collected 24 hours post-infection, using FACS
flow cytometry sorting with a monoclonal antibody against the mir-302
expression maker. RGFP (Clontech). These isolated sells were grown in the
mirPS cell culture medium as aforementioned.

In Vitro Molecular Guidance of the mirPS Cell Differentiation

[0074] In absence of any treatment except the feeder-free mirPS culture
medium, xenograft implantation of the mirPS cells into the uterus or
peritoneal cavity, but not other tissues, of female pseudopregnant
immunocompromised SCID-beige mice could form teratoma-like primordial
tissues. The use of immunocompromised nude mice was to provide an in vivo
environment mimicking transplantation therapy. The pseudopregnant mice
were made by intraperitoneally injection of 1 IU human menopausal
gonadotrophin (HMG) for two days and then human chorionic gonadotrophin
(hCG) for one more day. For in vitro molecular guidance into
spermatogonia lineage, mirPS cells were maintained on
polyornithine/laminin-coated dishes in DMEM/F12 (1:1; high glucose)
medium supplemented with charcoal-stripped 10% FBS, 4 mM L-glutamine, 1
mM sodium pyruvate, 5 ng/ml activin and 50 ng/ml dihydrotestosterone
(DHT) for 12 hours, at 37° C. under 5% CO2. Then the cells
were trypsinized, washed with 1×PBS, and collected in four aliquots
of chilled Matrigel (100 μl each) and one aliquots of 100 μl
1×PBS. Immediately after that, we transplanted the cells into the
hind limb muscle, peritoneum, uterus, subcutaneous neck skin (with
Matrigel) and tail vein (with PBS) of 6-week-old athymic
immunocompromised SCID-beige nude mice. The mice were anesthetized with
diethyl ether during experimental processing. One week later,
spermatogonia-like cells were found only in the uterus area. For
fibroblast/differentiation, we followed the same procedure as shown
above, except using regular phenol red-free DMEM medium supplemented with
10% FBS, 4 mM L-glutamine; 1 mM sodium pyruvate, 5 ng/ml noggin and 100
ng/ml transforming growth factor beta1 (TGF-β1) for 6 hours before
xenotransplantation. Fibroblast-like cells were found in the uterus one
week later. For chondrocyte differentiation, we performed the same
procedure as before but using regular RPMI 1640 medium supplemented with
10% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate and 100 ng/ml bone
morphogenetic protein 4 (BMP4) for 6 hours. Chondrocyte-like cells were
found only in the liver area.

[0096] Construction of the SpRNAi-RGFP transgene. The SpRNAi-RGFP
transgene was generated as reported (1, 2, 3), consisting of three parts:
one artificial intron, namely SpRNAi, and two exons derived from a
mutated red fluorescent HcRed1 chromoprotein gene isolated from
Heteractis crispa, namely RGFP. Synthetic oligonucleotides used for
generating the SpRNAi intron were: sense phosphorylated 5'-GTAAGTGGTC
CGATCGTCGC GACGCGTGAT TACTAACTAT CAATATCTTA ATCCTGTCCC TTTTTTTTCC
ACAGTAGGAC CTTCGTGCA-3' and antisense 5'-TGCACGAAGG TCCTACTGTG GAAAAAAAAG
GGACAGGATT AAGATATTGA TAGTTAGTAA TGACGCGTCG CGACGATCGG ACCACTTAC-3'
(Sigma-Genosys, St. Louis, Mo.). The SpRNAi intron was formed by
hybridization of an equal mixture (1:1) of each sequence at 94° C.
for 2 min, at 70° C. for 10 min and then at 4° C. in
1×PCR buffer (e.g., 50 mM Tris-HCl, pH 9.2 at 25° C., 16 mM
(NH4)2SO4, 1.75 mM MgCl2). The hybridized SpRNAi
intron was purified with a microcon-30 filter (Amicon, Beverly, Mass.) in
10 μl of autoclaved ddH2O, and than digested with a DraII
restriction enzyme (10 U) at 37° C. for 4 hours. The digested
intron was collected with a new microcon-30 filter in 10 μl of
autoclaved ddH2O. Concurrently, two RGFP exon sequences were
generated by enzymatic cleavage with DraII in the 208th nucleotide (nt)
site of the HcRed1 gene (BD Biosciences, Palo Alto, Calif.) and the
5'-end exon fragment was further blunt-ended by T4 DNA polymerase (5 U).
After that, the SpRNAi-RGFP transgene was formed by ligation of the
SpRNAi intron and the two RGFP exons. We first mixed an equal mixture
(1:1:1) of the intron and exons and incubated the mixture in 1×PCR
buffer from 50° C. to 10° C. over a period of 1 hour. Then
T4 DNA ligase (20 U) and buffer (Roche Biochemicals, Indianapolis, Ind.)
were added into the mixture and the ligation was carried out at
12° C. for 12 hours. For cloning the full-length SpRNAi-RGFP
transgene, the ligated products (10 ng) were amplified by high-fidelity
PCR (Roche) with primers (sense 5'-CTCGAGCATG GTGAGCGGCC TGCTGAA-3' and
antisense 5'-dTCTAGAAGTT GGCCTTCTCG GGCAGGT-3') at 94° C. for 1
min, at 54° for 1 min and then at 68° C. for 2 min. for 25
cycles. The resulting PCR products were fractionated on a 2% agarose gel
and a ˜900 base-pair (bp) sequence was extracted and purified by a
gel extraction kit (Qiagen, Valencia, Calif.), following the
manufacturer's suggestion. The nucleotide composition of the SpRNAi-RGFP
transgene was confirmed by DNA sequencing.

[0097] Flow Cytometry assay. Cells were trypsinized, pelleted and fixed by
re-suspending in 1 ml of pre-chilled 70% methanol in PBS for 1 hour at
-20° C. The cells were pelleted and washed once with 1 ml of PBS.
The cells were pelleted again and resuspended in 1 ml of 1 mg/ml
propidium iodide, 0.5 mg/ml RNase in PBS for 30 min at 37° C.
Approximately 15,000 cells were then analyzed on a BD FACSCalibur flow
cytometer (San Jose, Calif.). Cell doublets were excluded by plotting
pulse width versus pulse area and gating on the single cells. The
collected data were analyzed using the software package Flowjo using the
"Watson Pragmatic" algorithm (4). The first (left) and second (right)
peaks of the flow cytometry charts represented the levels of resting
G0/G1 and mitotic M phase cell populations in the entire tested cell
population, respectively.

[0101] Bisulfite PCR and genomic DNA sequencing. Genomic DNAs from about
two million cells were isolated with a DNA isolation kit (Roche) and
divided into two aliquots. One of the DNA aliquot (2 μg) was digested
with a CCGG-cutting restriction enzyme, HpaII, and then assessed with 1%
agarose gel electrophoresis to determine genome-wide demethylation. The
other aliquot (2 μg) was used for PCR cloning the complete 9,400
base-pair (bp) 5'-regulatory region of the Oct3/4 promoter
(NT--007592 nucleotides 21992184-22001688), before and after
bisulfite modification. Bisulfite modification was performed with a
CpGenome DNA modification kit (Chemicon), according to the manufacturers'
suggestions. The treatment of bisulfite to DNA converted all unmethylated
cytosines to uracils while methylated cytosines remained as cytosines.
For example, unmethylated ACGT sites, but not methylated ACGT, were
changed into AUGT sites. PCR primers specific to the target Oct3/4
5'-promoter region before and after bisulfite modification had been
designed and tested in the Takahashi's report (6), including two forward
primers 5'-GAGGAGTTGA GGGTAGTGTG-3' (for bisulfite-modified DNAs) and
5'-GAGGAGCTGA GGGCACTGTG-3' (for non-modified DNAs) and one reverse
primer 5'-GTAGAAGTGC CTCTGCCTTC C-3'. For PCR cloning, the genomic DNAs
(50 ng), either bisulfite-treated or untreated, were first mixed with the
primers (total 150 pmole) in 1× PCR buffer, heated to 94° C.
for 4 min, and immediately cooled on ice. After that, 25 cycles of PCR
were performed as follows: at 92° C. for 1 min, at 55° C.
for 1 min and then at 70° C. for 5 min, using a long template PCR
extension kit (Roche). The resulting products were collected with a PCR
purification kit (Qiagen) and 2 μg of the DNAs were digested with an
equal mixture (5 U each) of multiple ACGT-cutting restriction enzymes,
containing AclI (AACGTT), BmgBI (CACGTC), PmlI (CACGTG), SnaBI (TACGTA)
and HpyCH4IV (ACGT). Then the digested fragments were assessed using 3%
agarose gel electrophoresis. For DNA sequencing analysis, we further
amplified a 467-bp target region flanking the Oct3/4 transcription
initiation site (NT--007592 nucleotides 21996577-21997043), using
quantitative PCR (qPCR). Primers used were one forward primer
5'-GAGGCTGGAG TAGAAGGATT GCTTTGG-3' and one reverse primer 5'-CCCTCCTGAC
CGATCACCTC CACCACC-3'. The above PCR-cloned Oct3/4 5'promoter region (50
ng) were mixed with the primers (total 100 pmole) in 1× PCR buffer,
heated to 94° C. for 2 min, and immediately cooled on ice. Then,
20 cycles of PCR were performed as follows: at 94° C. for 30 sec
and at 68° C. for 1 min, using a high-fidelity PCR extension kit
(Roche). The amplified DNA products with a correct 467-bp size were
further fractionized by 3% agarose gel eleotrophoresis, purified with a
gel extraction kit (Qiagen), and then used in DNA sequencing. A detailed
profile of the DNA methylation sites was generated by comparing the
unchanged cytosines in the bisulfite-modified DNA to those in the
non-modified DNA sequence.

[0102] MicroRNA microarray analysis (p<0.01, n=3). At 70% confluency,
small RNAs from each cell culture were isolated, using the mirVANA®
miRNA isolation kit (Ambion) following the manufacturer's suggestion. The
purity and quantity of the isolated small RNAs were assessed, using 1%
formaldehyde-agarose gel electrophoresis, and spectrophotometer
measurement (Bio-Rad), and then immediately frozen in dry ice and
submitted to LC Sciences (San Diego, Calif.) for miRNA microarray
analysis. Each microarray chip was hybridized with a single sample
labeled with either Cy3 or Cy5 or a pair of samples labeled with Cy3 and
Cy5, respectively. Background subtraction and normalization were
performed. For a dual sample assay, a p-value calculation was performed
and a list of differentially expressed transcripts more than 3-fold was
produced. In the Cy3 and Cy5 intensity images (blue background), as
signal intensity increased from level 1 to level 65,535 the corresponding
color changed from blue to green, to yellow, and to red. The levels above
23,000 were considered to be positive calls in gene expression. In the
Cy5/Cy3 ratio image (black background), when Cy3 level was higher than
Cy5 level the color was green; when Cy3 level was equal to Cy5 level the
color was yellow; and when Cy5 level was higher than Cy3 level the color
was red.

[0103] Genome microarray analysts (p<0.01, n=4). Human genome GeneChip
U133A&B and plus 2.0 arrays (Affymetrix, Santa Clara, Calif.) containing
over 54,000 oligonucleotide probes were used to detect the expression
patterns of genome-wide 47,000 human gene transcripts in mirPS cells.
Total RNAs from each tested sample were isolated using RNeasy spin
columns (Qiagen). To prepare labeled probes for microarray hybridization,
the extracted total RNAs (2 μg) were converted into double-stranded
cDNAs with a synthetic oligo(dT)24-T7 promoter primer, 5'-GGCCAGTGAA
TTGTAATACG ACTCACTATA GGGAGGGGG-(dT)24-3', using Superscript Choice
system (Invitrogen). The resulting cDNAs were purified by
phenol/chloroform extractions, precipitated with ethanol, and resuspended
at a concentration of 0.5 μg/μl in diethyl pyrocarbonate
(DEPC)-treated ddH2O. Then, in vitro transcription was performed,
containing 1 μg of the dsDNAs, 7.5 mM unlabeled ATP and GTP, 5 mM
unlabeled UTP and CTP, and 2 mM biotin-labeled CTP and UTP
(biotin-11-CTP, biotin-16-UTP, Enzo Diagnostics), and 20 U of T7 RNA
polymerase. Reactions were carried out for 4 hours at 37° C. and
the resulting cRNAs were purified by RNeasy spin columns (Qiagen). A part
of the cRNA sample was separated on a 1% agarose gel to check the size
range, and then 10 μg of the cRNAs were fragmented randomly to an
average size of 50 bases by heating at 94° C. for 35 min in 40 mM
Tris-acetate, pH 8.0, 100 mM KOAc/30 mM MgOAc. Hybridizations were
completed in 200 μl of AFFY buffer (Affymetrix) at 40° C. for
16 hours with constant mixing. After hybridization, arrays were rinsed
three times with 200 μl of 6× SSPE-T buffer (1× 0.25 M
sodium chloride/15 mM sodium phosphate, pH 7.6/1 mM EDTA/0.005% Triton)
and then washed with 200 μl of 6× SSPE-T for 1 hour at
50° C. The arrays were further rinsed twice with 0.5× SSPE-T
and washed with 0.5× SSPE-T at 50° C. for 15 min. Then,
staining assays were done with 2 μg/ml streptavidinphycoerythrin
(Invitrogen-Molecular Probes) and 1 mg/ml acetylated BSA (Sigma) in
6× SSPET (pH 7.6). The arrays were read at 7.5 μm with a
confocal scanner (Molecular Dynamics). To identify the background
variations, we duplicated the microarray tests using the same sample and
selected two hundred genes, which were slightly presented in one side of
the tests, for further comparison. The samples were normalized using the
total average difference between perfectly matched probes and mismatched
probes. Then, alterations of overall genome-wide gene expression patterns
were analyzed using Affymetrix Microarray Suite version 5.0, Expression
Console® version 1.1.1 (Affymetrix) and Genesprings (Silicon Genetics)
softwares. Changes in gene expression rates more than 1-fold were
considered as positive differential genes. In gene clustering assays, as
signal intensity increased from level 1 to level 65,535 the corresponding
color changed from green to black, and to red. The level above 23,000 (in
red) was considered to be a positive call in individual gene expression.

[0104] After retroviral infection (˜12 hours), the medium is changed
to the mirPS cell medium as reported (Lin et al., 2008, RNA
14:2115-2124). Noggin may not be needed for Borne somatic cell types.
Three days later, positively infected cells are selected using FACS with
an antibody against RGFP (or any marker used for co-expression with the
mir-302 cluster). It takes about one or two more weeks to see small
colonies. If the mir-302 cluster is successfully expressed in the
infected cells, 95%-98% apoptotic tumor cells will be seen due to the
strong silencing of CDK2 and cyclin D1 and D2 (over 80% reduction) by
mir-302. Such strong apoptotic effect will not occur in mirPS cells
derived from normal somatic cells. As shown in Lin et al., 2008, RNA
14:2115-2124, CDK2 and cyclin D1 and D2, are valid targets for mir-302.
Therefore, only a small percentage of the cells survive after the
infection.

[0105] If outgrowth of prostate cancer PC3 or melanoma Colo-829 cells is
observed in the cultures, it is very likely that either the viral titer
used is too low or the mir-302 is not properly expressed, or both. The
retroviral titer used is preferably over MOI 30. The higher, the better;
however, if it is too high, all the cells will be arrested at the G1
phase. Thus, the concentration should be optimized for the cell
condition. Preferably, the Pol III- or CMV-driven siRNA/shRNA direct
expression systems are not employed due to their low expression rates in
Colo/PC3 cells (˜16 fold increase in total). This is probably due
to the short template and highly structured conformation of the mir-302
cluster (˜350 bp) which may be difficult to be, directly
transcribed. Preferably, mir-302 is not expressed with an eGFP marker,
which exhibits certain toxicity at a high concentration.

[0106] After colonies are observed (one to two weeks after FACS
selection), each colony should be isolated into a different 98-well plate
for continued growth for up to one month. The colonies will form
blastula-like embryoid bodies (EB). Up to this stage, they can be either
used for assays or dissociated for sub-culturing. See the Figures for
mirPS-Colo cell growth (similar to mirPC3). Early-stage EB should be used
for the experiments. The mirPS cells after the mature-stage EB may
contain some apoptotic cells in the center of the EB.